PMCC PMCC

Search tips
Search criteria

Advanced
Results 1-25 (77)
 

Clipboard (0)
None

Select a Filter Below

Journals
more »
Year of Publication
more »
1.  Mismatches Improve the Performance of Strand-Displacement Nucleic Acid Circuits** 
Catalytic hairpin assembly (CHA) has previously proven useful as a transduction and amplification method for nucleic acid detection. However, the two hairpin substrates in a CHA circuit can potentially react non-specifically even in the absence of a singles-stranded catalyst, and this non-specific background degrades signal-to-noise. The introduction of mismatched base-pairs that impede uncatalyzed strand exchange reactions greatly decreased background signal while only partially damping signal in the presence of catalyst. Various types and lengths of mismatches were assayed by fluorimetry and in many instances our MismatCHA designs yielded 100-fold signal-to-background ratios compared to a similar ratio of 4 with the perfectly matched substrates. These observations may prove to be of general utility for the design of non-enzymatic nucleic acid circuits.
doi:10.1002/anie.201307418
PMCID: PMC3983710  PMID: 24402831
Catalyzed hairpin assembly; Mismatch; Signal: background ratio
2.  Novel Modifications on C-terminal Domain of RNA Polymerase II can Fine-tune the Phosphatase Activity of Ssu72 
ACS chemical biology  2013;8(9):2042-2052.
The C-terminal domain of RNA polymerase II (CTD) modulates the process of transcription through sequential phosphorylation/dephosphorylation of its heptide repeats through which it recruits various transcription regulators. Ssu72 is the first characterized cis-specific CTD phosphatase that dephosphorylates Ser5 with a requirement for the adjacent Pro6 in a cis conformation. The recent discovery of Thr4 phosphorylation in the CTD calls into question whether such a modification can interfere with Ssu72 binding via the elimination of a conserved intra-molecular hydrogen bond in the CTD that is potentially essential for recognition. To test if Thr4 phosphorylation will abolish Ser5 dephosphorylation by Ssu72, we determined the kinetic and structural properties of Drosophila Ssu72-symplekin in complex with the CTD peptide with consecutive phosphorylated Thr4 and Ser5. Our mass spectrometric and kinetic data established that Ssu72 doesn’t dephosphorylate Thr4, but the existence of phosphoryl-Thr4 next to Ser5 reduces the activity of Ssu72 towards the CTD peptide by four fold. To our surprise, even though the intra-molecular hydrogen bond is eliminated due to the phosphorylation of Thr4, the CTD adopts an almost identical conformation to be recognized by Ssu72 with Ser5 phosphorylated alone or both Thr4/Ser5 phosphorylated. Our results indicate that Thr4 phosphorylation will not abolish the essential Ssu72 activity, which is needed for cell survival. Instead, the phosphatase activity of Ssu72 is fine-tuned by Thr4 phosphorylation and eventually may lead to changes in transcription. Overall, we report the first case of structural and kinetic effects of phosphorylated Thr4 on CTD modifying enzymes. Our results support a model in which a combinatorial cascade of CTD modification can modulate transcription.
doi:10.1021/cb400229c
PMCID: PMC4296584  PMID: 23844594
Ssu72 phosphatase; phosphorylation of RNA polymerase II; transcription regulation; CTD code; post-translational modification; intra-molecular hydrogen bond; x-ray crystallography
3.  G-quadruplex-generating PCR for visual colorimetric detection of amplicons 
Analytical biochemistry  2013;445:38-40.
We have developed a self-reporting PCR system for visual colorimetric gene detection and distinction of single nucleotide polymorphisms (SNP). Amplification is performed using target-specific primers modified with a 5’-end tail that is complementary to a G-quadruplex deoxyribozyme-forming sequence. At end-point G-quadruplexes are forced to fold from PCR-generated duplex DNA and then used to colorimetrically report the successful occurrence of PCR by assaying their peroxidase activity using a chromogenic substrate. Furthermore, primer design considerations for the G-quadruplex-generating PCR system have allowed us to visually distinguish SNPs associated with Mycobacterium tuberculosis drug resistance alleles.
doi:10.1016/j.ab.2013.10.010
PMCID: PMC3893031  PMID: 24135653
G-quadruplex; single nucleotide polymorphism; Mycobacterium tuberculosis; polymerase chain reaction; colorimetric detection
4.  Library Generation by Gene Shuffling 
This unit describes the process of gene shuffling (also known as sexual PCR). Gene shuffling is a facile method for the generation of sequence libraries containing the information from a family of related genes. Essentially, related genes are fragmented by DNase I digestion and reassembled by primerless PCR. The resulting chimeric genes can then be screened or selected for a desired function.
doi:10.1002/0471142727.mb1512s105
PMCID: PMC3983707  PMID: 24510437
directed evolution; recombination; PCR
5.  Real-time detection of isothermal amplification reactions with thermostable catalytic hairpin assembly 
Catalytic hairpin assembly (CHA) is an enzyme-free amplification method that has previously proven useful in amplifying and transducing signals at the terminus of nucleic acid amplification reactions. Here, for the first time, we engineered CHA be thermostable from 37 °C to 60 °C and in consequence have generalized its application to real-time detection of isothermal amplification reactions. CHA circuits were designed and optimized for both high and low temperature rolling circle amplification (RCA) and strand displacement amplification (SDA). The resulting circuits not only increased the specificity of detection, they also improved sensitivity by as much as 25- to 10,000-fold over comparable real-time detection methods. These methods have been condensed into a set of general rules for the design of thermostable CHA circuits with high signals and low noise.
doi:10.1021/ja4023978
PMCID: PMC3724415  PMID: 23647466
6.  DNA detection using origami paper analytical devices 
Analytical chemistry  2013;85(20):10.1021/ac402118a.
We demonstrate the hybridization-induced fluorescence detection of DNA on an origami-based paper analytical device (oPAD). The paper substrate was patterned by wax printing and controlled heating to construct hydrophilic channels and hydrophobic barriers in a three-dimensional fashion. A competitive assay was developed where the analyte, a single-stranded DNA (ssDNA), and a quencher-labeled ssDNA competed for hybridization with a fluorophore-labeled ssDNA probe. Upon hybridization of the analyte with the fluorophore-labeled ssDNA, a linear response of fluorescence vs. analyte concentration was observed with an extrapolated limit of detection < 5 nM and a sensitivity relative standard deviation as low as 3%. The oPAD setup was also tested against OR/AND logic gates, proving to be successful in both detection systems.
doi:10.1021/ac402118a
PMCID: PMC3852662  PMID: 24070108
Paper-based microfluidics; origami; DNA; fluorescence; logic gate; strand-displacement
7.  Adapting Enzyme-Free DNA Circuits to the Detection of Loop-Mediated Isothermal Amplification Reactions 
Analytical chemistry  2012;84(19):8371-8377.
Loop-mediated isothermal amplification of DNA (LAMP) is a powerful isothermal nucleic acid amplification technique that can accumulate ~109 copies from less than 10 copies of input template within an hour or two. Unfortunately, while the amplification reactions are extremely powerful, the quantitative detection of LAMP products is still analytically difficult. In this article, in order to both improve the specificity of LAMP detection and to make direct readout of LAMP amplification simpler and much more reliable, we have developed a non-enzymatic nucleic acid circuit (catalyzed hairpin assembly, CHA) that can both amplify and integrate the specific sequence signals present in LAMP amplicons. Through a hairpin acceptor, one of the four loop products amplified from the LAMP is transduced to an active catalyst ssDNA which can in turn trigger a CHA reaction. After CHA detection, even less than 10 molecules/μL model templates (M13mp18) can produce significant signal, and both non-specific template and parasitic amplicons cannot bring interference at all. More importantly, to further enhance the specificity, we have designed a dual-CHA circuit that only gave positive responses in presence of two LAMP loops. The AND-GATE detector will act as a simultaneous, specific readout of the LAMP product, rather than of competing and parasitic amplicons.
doi:10.1021/ac301944v
PMCID: PMC3478682  PMID: 22947054
LAMP; hairpin assembly; DNA circuit; amplification
8.  Bacteriophages use an expanded genetic code on evolutionary paths to higher fitness 
Nature chemical biology  2014;10(3):178-180.
Bioengineering advances have made it possible to fundamentally alter the genetic codes of organisms. However, the evolutionary consequences of expanding an organism's genetic code with a non-canonical amino acid are poorly understood. Here we show that bacteriophages evolved on a host that incorporates 3-iodotyrosine at the amber stop codon acquired neutral and beneficial mutations to this new amino acid in their proteins, demonstrating that an expanded genetic code increases evolvability.
doi:10.1038/nchembio.1450
PMCID: PMC3932624  PMID: 24487692
9.  Proliferation and Migration of Tumor Cells in Tapered Channels 
Biomedical microdevices  2013;15(4):635-643.
Tumor cells depict two deviant tendencies; over-proliferation and vigorous migration. A tapered channel device is designed and fabricated for in vitro studying inhibited proliferation and migration of human glioblastoma (hGBM) cells when exposed to a novel aptamer targeting epidermal growth factor receptors (EGFR). The device is integrated with controlled ambient and microscope for providing real-time and quantitative characterization of the tumor cell behavior in vitro. The results show that hGBM cells loose proliferation and motility when exposed to the anti-EGFR aptamers. The aptamer directly inhibits and blocks EGF-induced EGFR phosphorylation. This also reduces the ability of cells to remodel their internal structure for invasion through narrow constrictions. This provides a framework for possible studies on efficacy of other inhibiting molecules.
doi:10.1007/s10544-012-9721-0
PMCID: PMC3578089  PMID: 23104156
10.  A ‘resource allocator’ for transcription based on a highly fragmented T7 RNA polymerase 
Molecular Systems Biology  2014;10(7):742.
Synthetic genetic systems share resources with the host, including machinery for transcription and translation. Phage RNA polymerases (RNAPs) decouple transcription from the host and generate high expression. However, they can exhibit toxicity and lack accessory proteins (σ factors and activators) that enable switching between different promoters and modulation of activity. Here, we show that T7 RNAP (883 amino acids) can be divided into four fragments that have to be co-expressed to function. The DNA-binding loop is encoded in a C-terminal 285-aa ‘σ fragment’, and fragments with different specificity can direct the remaining 601-aa ‘core fragment’ to different promoters. Using these parts, we have built a resource allocator that sets the core fragment concentration, which is then shared by multiple σ fragments. Adjusting the concentration of the core fragment sets the maximum transcriptional capacity available to a synthetic system. Further, positive and negative regulation is implemented using a 67-aa N-terminal ‘α fragment’ and a null (inactivated) σ fragment, respectively. The α fragment can be fused to recombinant proteins to make promoters responsive to their levels. These parts provide a toolbox to allocate transcriptional resources via different schemes, which we demonstrate by building a system which adjusts promoter activity to compensate for the difference in copy number of two plasmids.
doi:10.15252/msb.20145299
PMCID: PMC4299498  PMID: 25080493
genetic circuit; resource allocation; split protein; synthetic biology; T7 RNA polymerase
11.  Dynamic Reorganization of Metabolic Enzymes into Intracellular Bodies 
Both focused and large-scale cell biological and biochemical studies have revealed that hundreds of metabolic enzymes across diverse organisms form large intracellular bodies. These proteinaceous bodies range in form from fibers and intracellular foci—such as those formed by enzymes of nitrogen and carbon utilization and of nucleotide biosynthesis—to high-density packings inside bacterial microcompartments and eukaryotic microbodies. Although many enzymes clearly form functional mega-assemblies, it is not yet clear for many recently discovered cases whether they represent functional entities, storage bodies, or aggregates. In this article, we survey intracellular protein bodies formed by metabolic enzymes, asking when and why such bodies form and what their formation implies for the functionality—and dysfunctionality—of the enzymes that comprise them. The panoply of intracellular protein bodies also raises interesting questions regarding their evolution and maintenance within cells. We speculate on models for how such structures form in the first place and why they may be inevitable.
doi:10.1146/annurev-cellbio-101011-155841
PMCID: PMC4089986  PMID: 23057741
self-assembly; allosteric regulation; fibers; foci; storage bodies; aggregates; metabolic efficiency
12.  Structure-based non-canonical amino acid design to covalently crosslink an antibody–antigen complex 
Journal of structural biology  2013;185(2):215-222.
Engineering antibodies to utilize non-canonical amino acids (NCAA) should greatly expand the utility of an already important biological reagent. In particular, introducing crosslinking reagents into antibody complementarity determining regions (CDRs) should provide a means to covalently crosslink residues at the antibody–antigen interface. Unfortunately, finding the optimum position for crosslinking two proteins is often a matter of iterative guessing, even when the interface is known in atomic detail. Computer-aided antibody design can potentially greatly restrict the number of variants that must be explored in order to identify successful crosslinking sites. We have therefore used Rosetta to guide the introduction of an oxidizable crosslinking NCAA, l-3,4-dihydroxyphenylalanine (l-DOPA), into the CDRs of the anti-protective antigen scFv antibody M18, and have measured crosslinking to its cognate antigen, domain 4 of the anthrax protective antigen. Computed crosslinking distance, solvent accessibility, and interface energetics were three factors considered that could impact the efficiency of l-DOPA-mediated crosslinking. In the end, 10 variants were synthesized, and crosslinking efficiencies were generally 10% or higher, with the best variant crosslinking to 52% of the available antigen. The results suggest that computational analysis can be used in a pipeline for engineering crosslinking antibodies. The rules learned from l-DOPA crosslinking of antibodies may also be generalizable to the formation of other crosslinked interfaces and complexes.
doi:10.1016/j.jsb.2013.05.003
PMCID: PMC4086636  PMID: 23680795
Computer-aided design; Structure-based design; Rosetta; Antibody; Non-canonical amino acid; Crosslinking; Binding affinity; l-DOPA
13.  Exquisite allele discrimination by toehold hairpin primers 
Nucleic Acids Research  2014;42(15):e120.
The ability to detect and monitor single nucleotide polymorphisms (SNPs) in biological samples is an enabling research and clinical tool. We have developed a surprising, inexpensive primer design method that provides exquisite discrimination between SNPs. The field of DNA computation is largely reliant on using so-called toeholds to initiate strand displacement reactions, leading to the execution of kinetically trapped circuits. We have now similarly found that the short toehold sequence to a target of interest can initiate both strand displacement within the hairpin and extension of the primer by a polymerase, both of which will further stabilize the primer:template complex. However, if the short toehold does not bind, neither of these events can readily occur and thus amplification should not occur. Toehold hairpin primers were used to detect drug resistance alleles in two genes, rpoB and katG, in the Mycobacterium tuberculosis genome, and ten alleles in the Escherichia coli genome. During real-time PCR, the primers discriminate between mismatched templates with Cq delays that are frequently so large that the presence or absence of mismatches is essentially a ‘yes/no’ answer.
doi:10.1093/nar/gku558
PMCID: PMC4150758  PMID: 24990378
14.  In Vitro Selection Using Modified or Unnatural Nucleotides 
Incorporation of modified nucleotides into in vitro RNA or DNA selections offer many potential advantages, such as the increased stability of selected nucleic acids against nuclease degradation, improved affinities, expanded chemical functionality, and increased library diversity. This unit provides useful information and protocols for in vitro selection using modified nucleotides. It includes a discussion of when to use modified nucleotides; protocols for evaluating and optimizing transcription reactions, as well as confirming the incorporation of the modified nucleotides; protocols for evaluating modified nucleotide transcripts as template in reverse transcription reactions; protocols for the evaluation of the fidelity of modified nucleotides in the replication and the regeneration of the pool; and a protocol to compare modified nucleotide pools and selection conditions.
doi:10.1002/0471142700.nc0906s07
PMCID: PMC4068349  PMID: 18428900
15.  Pattern Transformation with DNA Circuits 
Nature chemistry  2013;5(12):1000-1005.
Readily programmable chemical networks are important tools as the scope of chemistry expands from individual molecules to larger molecular systems. While many complex systems have been constructed using conventional organic and inorganic chemistry, the programmability of biological molecules such as nucleic acids allows for precise, high-throughput, and automated design, as well as simple, rapid, and robust implementation. Here we show that systematic and quantitative control over the diffusivity and reactivity of DNA molecules yields highly programmable chemical reaction networks (CRNs) that execute at the macroscale. In particular, we design and implement non-enzymatic DNA circuits capable of performing pattern transformation algorithms such as edge detection. We also show that it is possible to fine-tune and multiplex such circuits. We believe these strategies will provide programmable platforms for prototyping CRNs, for discovering bottom-up construction principles, and for generating patterns in materials.
doi:10.1038/nchem.1764
PMCID: PMC3970425  PMID: 24256862
16.  Analyzing machupo virus-receptor binding by molecular dynamics simulations 
PeerJ  2014;2:e266.
In many biological applications, we would like to be able to computationally predict mutational effects on affinity in protein–protein interactions. However, many commonly used methods to predict these effects perform poorly in important test cases. In particular, the effects of multiple mutations, non alanine substitutions, and flexible loops are difficult to predict with available tools and protocols. We present here an existing method applied in a novel way to a new test case; we interrogate affinity differences resulting from mutations in a host–virus protein–protein interface. We use steered molecular dynamics (SMD) to computationally pull the machupo virus (MACV) spike glycoprotein (GP1) away from the human transferrin receptor (hTfR1). We then approximate affinity using the maximum applied force of separation and the area under the force-versus-distance curve. We find, even without the rigor and planning required for free energy calculations, that these quantities can provide novel biophysical insight into the GP1/hTfR1 interaction. First, with no prior knowledge of the system we can differentiate among wild type and mutant complexes. Moreover, we show that this simple SMD scheme correlates well with relative free energy differences computed via free energy perturbation. Second, although the static co-crystal structure shows two large hydrogen-bonding networks in the GP1/hTfR1 interface, our simulations indicate that one of them may not be important for tight binding. Third, one viral site known to be critical for infection may mark an important evolutionary suppressor site for infection-resistant hTfR1 mutants. Finally, our approach provides a framework to compare the effects of multiple mutations, individually and jointly, on protein–protein interactions.
doi:10.7717/peerj.266
PMCID: PMC3940602  PMID: 24624315
Arenavirus; Machupo; Protein–protein interaction; Molecular dynamics; Computational mutagenesis; Free energy perturbation
17.  High-throughput sequencing of the paired human immunoglobulin heavy and light chain repertoire 
Nature biotechnology  2013;31(2):166-169.
Each B-cell receptor consists of a pair of heavy and light chains. High-throughput sequencing can identify large numbers of heavy- and light-chain variable regions (VH and VL) in a given B-cell repertoire, but information about endogenous pairing of heavy and light chains is lost after bulk lysis of B-cell populations. Here we describe a way to retain this pairing information. In our approach, single B cells (>5 × 104 capacity per experiment) are deposited in a high-density microwell plate (125 pl/well) and lysed in situ. mRNA is then captured on magnetic beads, reverse transcribed and amplified by emulsion VH:VL linkage PCR. The linked transcripts are analyzed by Illumina high-throughput sequencing. We validated the fidelity of VH:VL pairs identified by this approach and used the method to sequence the repertoire of three human cell subsets—peripheral blood IgG+ B cells, peripheral plasmablasts isolated after tetanus toxoid immunization and memory B cells isolated after seasonal influenza vaccination.
doi:10.1038/nbt.2492
PMCID: PMC3910347  PMID: 23334449
18.  Design and application of cotranscriptional non-enzymatic RNA circuits and signal transducers 
Nucleic Acids Research  2014;42(7):e58.
Nucleic acid circuits are finding increasing real-life applications in diagnostics and synthetic biology. Although DNA has been the main operator in most nucleic acid circuits, transcriptionally produced RNA circuits could provide powerful alternatives for reagent production and their use in cells. Towards these goals, we have implemented a particular nucleic acid circuit, catalytic hairpin assembly, using RNA for both information storage and processing. Our results demonstrated that the design principles developed for DNA circuits could be readily translated to engineering RNA circuits that operated with similar kinetics and sensitivities of detection. Not only could purified RNA hairpins perform amplification reactions but RNA hairpins transcribed in vitro also mediated amplification, even without purification. Moreover, we could read the results of the non-enzymatic amplification reactions using a fluorescent RNA aptamer ‘Spinach’ that was engineered to undergo sequence-specific conformational changes. These advances were applied to the end-point and real-time detection of the isothermal strand displacement amplification reaction that produces single-stranded DNAs as part of its amplification cycle. We were also able to readily engineer gate structures with RNA similar to those that have previously formed the basis of DNA circuit computations. Taken together, these results validate an entirely new chemistry for the implementation of nucleic acid circuits.
doi:10.1093/nar/gku074
PMCID: PMC3985647  PMID: 24493736
19.  Biotechnological applications of mobile group II introns and their reverse transcriptases: gene targeting, RNA-seq, and non-coding RNA analysis 
Mobile DNA  2014;5:2.
Mobile group II introns are bacterial retrotransposons that combine the activities of an autocatalytic intron RNA (a ribozyme) and an intron-encoded reverse transcriptase to insert site-specifically into DNA. They recognize DNA target sites largely by base pairing of sequences within the intron RNA and achieve high DNA target specificity by using the ribozyme active site to couple correct base pairing to RNA-catalyzed intron integration. Algorithms have been developed to program the DNA target site specificity of several mobile group II introns, allowing them to be made into ‘targetrons.’ Targetrons function for gene targeting in a wide variety of bacteria and typically integrate at efficiencies high enough to be screened easily by colony PCR, without the need for selectable markers. Targetrons have found wide application in microbiological research, enabling gene targeting and genetic engineering of bacteria that had been intractable to other methods. Recently, a thermostable targetron has been developed for use in bacterial thermophiles, and new methods have been developed for using targetrons to position recombinase recognition sites, enabling large-scale genome-editing operations, such as deletions, inversions, insertions, and ‘cut-and-pastes’ (that is, translocation of large DNA segments), in a wide range of bacteria at high efficiency. Using targetrons in eukaryotes presents challenges due to the difficulties of nuclear localization and sub-optimal magnesium concentrations, although supplementation with magnesium can increase integration efficiency, and directed evolution is being employed to overcome these barriers. Finally, spurred by new methods for expressing group II intron reverse transcriptases that yield large amounts of highly active protein, thermostable group II intron reverse transcriptases from bacterial thermophiles are being used as research tools for a variety of applications, including qRT-PCR and next-generation RNA sequencing (RNA-seq). The high processivity and fidelity of group II intron reverse transcriptases along with their novel template-switching activity, which can directly link RNA-seq adaptor sequences to cDNAs during reverse transcription, open new approaches for RNA-seq and the identification and profiling of non-coding RNAs, with potentially wide applications in research and biotechnology.
doi:10.1186/1759-8753-5-2
PMCID: PMC3898094  PMID: 24410776
Genome engineering; Metabolic engineering; Next-generation RNA sequencing; Ribozyme; Synthetic biology; Systems biology; Targetron
20.  Capture, Isolation and Release of Cancer Cells with Aptamer-functionalized Glass Bead Array 
Lab on a chip  2012;12(22):4693-4701.
Early detection and isolation of circulating tumor cells (CTC) can enable better prognosis for cancer patients. A Hele-Shaw device with aptamer functionalized glass beads is designed, modeled, and fabricated to efficiently isolate cancer cells from a cellular mixture. The glass beads are functionalized with anti-epidermal growth factor receptor (EGFR) aptamer and sit in ordered array of pits in PDMS channel. A PDMS encapsulation is then used to cover the channel and flow through cell solution. The beads capture cancer cells from flowing solution depicting high selectivity. The cell-bound glass beads are then re-suspended from the device surface followed by the release of 92% cells from glass beads using combination of soft shaking and anti-sense RNA. This approach ensures that the cells remain in native state and undisturbed during capture, isolation and elution for post-analysis. The use of highly selective anti-EGFR aptamer with the glass beads in an array and subsequent release of cells with antisense molecules provide multiple levels of binding and release opportunities that can help in defining new classes of CTC enumeration devices.
doi:10.1039/c2lc21251j
PMCID: PMC3498495  PMID: 22983436
21.  Directed evolution of gold nanoparticle delivery to cells 
Chemical communications (Cambridge, England)  2009;46(3):10.1039/b920865h.
A newly selected anti-receptor (anti-EGFR) aptamer was conjugated to gold nanoparticles via a facile hybridization method and was found to specifically and quantitatively direct the delivery of gold nanoparticles to cells expressing EGFR through receptor-mediated endocytosis.
doi:10.1039/b920865h
PMCID: PMC3826538  PMID: 20066302
22.  Aptamer-Mediated Delivery of Chemotherapy to Pancreatic Cancer Cells 
Nucleic Acid Therapeutics  2012;22(5):295-305.
Gemcitabine is a nucleoside analog that is currently the best available single-agent chemotherapeutic drug for pancreatic cancer. However, efficacy is limited by our inability to deliver sufficient active metabolite into cancer cells without toxic effects on normal tissues. Targeted delivery of gemcitabine into cancer cells could maximize effectiveness and concurrently minimize toxic side effects by reducing uptake into normal cells. Most pancreatic cancers overexpress epidermal growth factor receptor (EGFR), a trans-membrane receptor tyrosine kinase. We utilized a nuclease resistant RNA aptamer that binds and is internalized by EGFR on pancreatic cancer cells to deliver gemcitabine-containing polymers into EGFR-expressing cells and inhibit cell proliferation in vitro. This approach to cell type–specific therapy can be adapted to other targets and to other types of therapeutic cargo.
doi:10.1089/nat.2012.0353
PMCID: PMC3464421  PMID: 23030589
23.  Analysis of DNA-Guided Self Assembly of Microspheres Using Imaging Flow Cytometry 
Journal of the American Chemical Society  2012;134(37):15245-15248.
Imaging flow cytometry was used to analyze the self assembly of DNA-conjugated polystyrene microspheres. This technique enables quantitative analysis of the assembly process and thereby enables detailed analysis of effect of structural and process variables on the yield of assembly. In a demonstration of the potential of this technique, the influence of DNA strands base pair (bp) length was examined and it was found that 50 bp was sufficient to efficiently drive the assembly of microspheres, forming not only dimers but also chain-like structures. The effect of stoichiometry on yield was also examined. The analysis demonstrated that self assembly of 50 bp microspheres can be driven to near completion by stoichiometric excess in a manner similar to Le Chatelier’s principle in common chemical equilibrium.
doi:10.1021/ja3066896
PMCID: PMC3470448  PMID: 22938015
Self Assembly; DNA; Flow Cytometry
24.  Spatial Control of DNA Reaction Networks by DNA Sequence 
Molecules (Basel, Switzerland)  2012;17(11):13390-13402.
We have developed a set of DNA circuits that execute during gel electrophoresis to yield immobile, fluorescent features in the gel. The parallel execution of orthogonal circuits led to the simultaneous production of different fluorescent lines at different positions in the gel. The positions of the lines could be rationally manipulated by changing the mobilities of the reactants. The ability to program at the nanoscale so as to produce patterns at the macroscale is a step towards programmable, synthetic chemical systems for generating defined spatiotemporal patterns.
doi:10.3390/molecules171113390
PMCID: PMC3764599  PMID: 23143151
reaction-diffusion; electrophoresis; chemical reaction networks; DNA circuits; strand displacement reactions
25.  Generalized bacterial genome editing using mobile group II introns and Cre-lox 
A general bacterial genome engineering framework, ‘Genome Editing via Targetrons and Recombinases' (GETR), is presented. GETR combines mobile group II introns (targetrons) and the Cre/lox system to allow genomic manipulations at a large scale.
The combination of targetrons and Cre/lox represents a broad-host range solution to genome editing.Engineered targetrons were used to deliver lox sites site-specifically into the bacterial genome.Targetrons carrying lox sites were used to generate large-scale insertions, deletions, inversions, and unique cut-and-paste operations in bacterial genomes.
Efficient bacterial genetic engineering approaches with broad-host applicability are rare. We combine two systems, mobile group II introns (‘targetrons') and Cre/lox, which function efficiently in many different organisms, into a versatile platform we call GETR (Genome Editing via Targetrons and Recombinases). The introns deliver lox sites to specific genomic loci, enabling genomic manipulations. Efficiency is enhanced by adding flexibility to the RNA hairpins formed by the lox sites. We use the system for insertions, deletions, inversions, and one-step cut-and-paste operations. We demonstrate insertion of a 12-kb polyketide synthase operon into the lacZ gene of Escherichia coli, multiple simultaneous and sequential deletions of up to 120 kb in E. coli and Staphylococcus aureus, inversions of up to 1.2 Mb in E. coli and Bacillus subtilis, and one-step cut-and-pastes for translocating 120 kb of genomic sequence to a site 1.5 Mb away. We also demonstrate the simultaneous delivery of lox sites into multiple loci in the Shewanella oneidensis genome. No selectable markers need to be placed in the genome, and the efficiency of Cre-mediated manipulations typically approaches 100%.
doi:10.1038/msb.2013.41
PMCID: PMC3792343  PMID: 24002656
bacterial genome engineering; Cre-lox; mobile group II introns; Staphylococcus aureus; Shewanella oneidensis

Results 1-25 (77)